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A cryopreservation protocol based on air dehydration of explants placed on aluminium cryo-plates, termed D cryo-plate, was successfully developed for in vitro mat rush (Juncus decipiens Nakai) lateral buds. The buds of line 'Hiroshima 4gou(1)' with basal stems were dissected from multiple shoots and precultured overnight at 25°C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates, each one with 10 elliptical wells (2.5 mm long, 1.5 mm wide and 0.75 mm deep) and embedded in calcium alginate gel with 0.4 M sucrose and 1 M glycerol. Osmoprotection was performed by immersing the cryo-plates with buds for 30 min at 25°C in loading solution (2 M glycerol+1.0 M sucrose). Buds were dehydrated to 26% moisture content (fresh weight) by placing the cryo-plates for 2.5 h either in the air current of a laminar flow cabinet or in Petri dishes containing silica gel. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in liquid medium with 1.0 M sucrose for 15 min at room temperature. Under these conditions, regrowth of cryopreserved buds of line, 'Hiroshima 4gou (1)' was 93% after four weeks culture. The average regrowth of 20 mat rush lines was 88%. The D cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1-1.5 mmϫ1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryoplates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation. (LS) and PVS2 without floating and/or clinging of shoot tips and also with no or low level of injury to those shoot tips. In this paper, this V-Cryo-plate procedure was applied for the cryopreservation of carnation.The carnation plants used in this study were obtained from the Nishinihon Station, NCSS, sub-bank of NIAS Genebank Project, in Japan in April 2010. Four cultivars of carnation, 'Melissa', 'Grana', 'Terra Cotta' and 'Yell', were tested. Apical shoots, excised from in vivo explants, were surface-sterilized successively with 70% ethanol for 1 min, and 0.5% sodium hypochlorite for 10 min, and then rinsed twice with sterilized distilled water. After surface sterilization, shoot tips with basal plates (1 mm longϫ1 mm wide) were excised from the shoots and cultured on 70 ml of 0.4% (w/v) gellan gum-solidified MS medium (Murashige, Skoog 1962) in a culture flask (80 mmϫ100 mm) containing 1 mg l Ϫ1 thidiazuron, 1 mg l Ϫ1 naphthalene acetic acid and 3% (w/v) sucrose (Jose et al. 2010). After 50 days of culture, shoots were transferred onto gellan gum-solidified MS medium devoid of plant growth regulators. Then stock plants were subcultured every two months on MS medium. Cultures were incubated at 25°C with a 16 h photoperiod under white fluorescent light (52 mmol m Ϫ2 s Ϫ1) in the culture flask (standard condition). For cryopreservation of in vitro shoot tips of carnation, the V-Cryo-plate procedure was applied. The size of the aluminum cryoplate used was 7 mmϫ37 mmϫ0.5 mm with ten wells (diameter 1.5 mm, depth 0.75 mm) ( Figure 1B). These plates fit in 2 ml cryotube. The different steps of the VCryo-plate procedure are as follows; 1) Cut shoots (5 mm) with a lateral bud and plate on solidified MS medium and culture for 2 weeks at 25°C in standard conditions ( Figure 1A). Then, dissect shoot tips with basal plate (1-1.5 mm longϫ1 mm wide) from the shoots and preculture for 2 days at 25°C on the MS medium with 0.3 M sucrose. 2) Place an aluminum cryo-plate in Petri-dish and pour 2.0-2.5 ml 2% (w/v) Na-alginate solution with 0.4 M sucrose in calcium-free M...

Cryopreservation using an aluminium cryo-plate was successfully applied to in vitro-grown strawberry (Fragaria £ ananassa Duch.) shoot tips. The shoots were cold-hardened at 58C for 3 weeks with an 8-h photoperiod. The shoot tips (1.5 -2.0 mm £ 0.5-1.0 mm) were dissected from the shoot and pre-cultured at 58C for 2 d on Murashige and Skoog medium containing 2 M glycerol and 0.3 M sucrose. The pre-cultured shoot tips were placed on the aluminium cryo-plate containing ten wells embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in a loading solution (2 M glycerol and 0.8 M sucrose) for 30 min at 258C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 258C. Then, the cryo-plate with shoot tips was transferred into an uncapped cryotube that was held on a cryo-cane and directly immersed into liquid nitrogen (LN). After storage in LN, shoot tips attached to the cryo-plate were directly immersed into 2 ml of a 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 15 strawberry cultivars reached 81%. This new method has many advantages and will facilitate the cryostorage of strawberry germplasm.

Cryopreservation has become a very important tool for the long-term storage of plant germplasm. A new cryopreservation protocol based on air dehydration of explants placed on aluminum cryo-plates, termed the D cryo-plate technique, was developed. In this study, the most suitable conditions of cryopreservation for dormant shoot tips of Japanese persimmon (Diospyros kaki Thunb. 'Saijo') using the D cryo-plate technique were investigated. Dormant one-year-old shoots of persimmon were collected from the experimental farm of Shimane University in January 2013 and stored at 2°C until use. After surface sterilization, shoot tips of about 1 mm in size were dissected from the dormant buds and precultured overnight at 25°C on solidified 1/2MS medium containing 0.3 M sucrose. Precultured shoot tips were placed on aluminum cryo-plates and embedded in calcium alginate gel. Osmoprotection of shoot tips was performed by immersing the cryo-plates for 30 min at 25°C in an LS solution containing 2 M glycerol + 1.0 M sucrose in 1/2MS solution. For the D cryo-plate technique, encapsulated shoot tips were dehydrated by placing the cryo-plates in the air current of a laminar flow cabinet for 30-90 min. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, the cryo-plates were immersed in 1/2MS medium containing 1.0 M sucrose for 20 min at 25°C. In this study, the preculture did not improve the regrowth after cryopreservation; however, we consider that it should be performed in the D cryo-plate procedure for application of other cultivars and utilization in genebanks. A high regrowth rate of cryopreserved shoot tips (84%) was achieved after dehydration for 30 min. This optimized procedure was applied to 10 additional persimmon cultivars, resulting in regrowth rates ranging between 67 and 97%, with an average of 87%. As shoot tips derived from dormant buds proved to be highly tolerant to liquid nitrogen exposure, the D cryoplate technique may facilitate long-term conservation of persimmon germplasm.

Cryopreservation is an effective approach to conserve sugarcane germplasm for long term. This study was conducted to develop D cryo-plate technique to conserve sugarcane's shoot tips. This new and effective cryopreservation technique will serve the purpose to conserve sugarcane germplasm efficiently on the large scale at genebanks. Sugarcane variety Ni-1 were used to optimize the protocol and after optimization 11 other varieties were tested with optimized protocol. It was observed that shoot tips of Ni-1 with a length of 1.5 to 2.0 mm, precultured on semi-solid 1/2 MS medium for 1 day and semi-solid MS medium with 0.3 M sucrose for 1 day, treated with loading solution containing 2.0 M glycerol + 1.2 M sucrose for 30 min and air dehydrated in an air laminar flow's air current for 45 min, removed from alginate gel after cryopreservation and kept in dark for seven days produced maximum regrowth (97.7%). Range of 20.0% to 100% with an average of 52.1 % was observed among sugarcane varieties for regrowth after cryopreservation with optimized D cryoplate protocol.

Bitter gourd is one of the important cucurbits and highly liked among both farmers and consumers due to its high net return and nutritional value. However, being monoecious, it exhibits substantial variation in flower bearing pattern. Plant growth regulators (PGRs) are known to influence crop phenology while gibberellic acid (GA3) is one of the most prominent PGRs that influence cucurbits phenology. Therefore, a field trial was conducted at University of Agriculture Faisalabad to evaluate the impact of a commercial product of gibberellic acid (GA3) on growth, yield and quality attributes of two bitter gourd (Momordica charantiaL.) cultivars. We used five different concentrations (0.4 g, 0.6 g, 0.8 g, 1.0 g, and 1.2 g per litre) of commercial GA3 product (Gibberex, 10% Gibberellic acid). Results showed that a higher concentration of gibberex (1.0 and 1.20 g L−1 water) enhanced the petiole length, intermodal length, and yield of bitter gourd cultivars over control in Golu hybrid and Faisalabad Long. A significant decrease in the enzyme superoxidase dismutase, peroxidase, and catalase activities were observed with an increasing concentration of gibberex (1.0 and 1.20 gL−1 water) as compared to control. These results indicate that the exogenous application of gibberex at a higher concentration (1.2 g L−1) has a dual action in bitter gourd plant: i) it enhances the plant growth and yield, and ii) it also influenced the antioxidant enzyme activities in fruits. These findings may have a meaningful, practical use for farmers involved in agriculture and horticulture.